PROJECT SUMMARY Estrogen synthesis is catalyzed by aromatase (product of the CYP19A1 gene). In humans, aromatase is expressed in placenta, gonads, brain, bone and adipose stromal cells and is upregulated in carcinoma of the breast and lung, endometriosis and uterine leiomyoma. Expression of aromatase in these tissues is controlled by unique tissue-specific promoters. Because of exciting new directions of our ongoing research, in the proposed studies, we will specifically focus our efforts on further defining the mechanisms that mediate tissuespecific expression and regulation of aromatase in placenta and breast cancer. In Specific Aim 1, cultured human trophoblasts will be used to investigate the genetic and epigenetic mechanisms whereby critical transcription factors and coregulators upregulate hCYP19 expression during human trophoblast differentiation and block its expression with hypoxia. In Specific Aim 2, we will test the hypothesis that the O2-dependent induction of hCYP19 in human placental cells is mediated, in part, by specific microRNAs (miRNAs/miRs) that regulate key transcription factors and coregulators. In view of the suggested role of hypoxia in the pathogenesis of preeclampsia, a disease of inadequate placental perfusion, the established role of estrogen in vasculogenesis, and our findings regarding O2-dependent regulation of human trophoblast differentiation and aromatase expression, we are hopeful that our studies will shed new light on the etiology of this devastating disease. In human breast cancer cells, we have found that cAMP and cytokine induction of hCYP19 and cyclooxygenase-2 (hCOX-2) expression is markedly inhibited by progesterone (P4)/progesterone receptor (PR). In studies outlined in Specific Aim 3, we will examine the transcriptional mechanisms whereby aromatase is upregulated in breast cancer and test the hypothesis that the inhibitory effects of PR on hCYP19 and hCOX-2 are PR-isoform-specific, associated with PR sumoylation and mediated by PR interaction with activating transcription factors bound to the hCYP19 and hCOX-2 promoters to inhibit their binding and/or activity, promote recruitment of corepressors and the closing of chromatin structure. Our hypothesis that P4/PR has anti-inflammatory and anti-proliferative effects in the breast will further be tested in mice with a mammary gland-specific disruption of the PR gene.